The long-term goal of the proposed studies is to understand the molecular mechanisms by which the Drosophila Polycomb-group (PcG) proteins maintain the transcriptional silence of target genes. Originally identified as negative regulators of the homeotic genes of the Antennapedia and bithorax gone complexes, PcG proteins are conserved in plants, worms and mammals and play important roles in cellular determination and memorization of cell fate. PcG proteins do not initiate the repression of target genes. Rather, following initial repression by short-lived transcription factors, PcG proteins recognize the repressed state and establish repressive chromatin domains that heritably maintain silence through many cell cycles. Most PcG proteins function as components of multimeric complexes. The polypeptide compositions of two PcG complexes have been described, and other complexes have been identified, but not yet purified. Heritable maintenance of transcriptional silence requires repeated and faithful recruitment of PcG complexes to target genes following mitosis, in the proposed studies, we will examine the role of a sequence-specific DNA binding PcG protein in recruitment of PcG complexes to target sites in proliferating cells. PcG-dependent silencing of target genes involves modification of chromatin structure. We will use RNA interference (RNAi) and chromatin immunoprecipitation (X-ChIP) techniques to examine the assembly of PcG proteins at target sites and the specific effects of individual PcG proteins and protein complexes on chromatin modification. Using this approach, the activity of a novel PcG protein has been identified. Reverse genetic studies will be performed to examine its in vivo function. A previously uncharacterized PcG complex will be biochemically identified using traditional chromatographic methods. The identity of its constituent polypeptides will be determined using mass spectrometry. Information gained from these complimentary experimental approaches will lead to better understanding of the molecular mechanisms by which PcG proteins maintain the transcriptional silence of target genes. [unreadable] [unreadable]